The initial Drosophila Genome Center grant proposed to build a clone-based physical map of the Drosophila melanogaster euchromatic genome using a bacteriophage P1 library and a non-random mapping strategy that relied on STS content mapping. The major motivation for constructing this map is to provide the directed genomic sequencing project at LBL with well- characterized clones from which to generate sequencing templates. Toward this goal we have established a production STS mapping effort at LBL capable of assigning more 100 STS markers per month to the physical map. The major source of STS markers for this project derive from the insert ends of P1 clones that have been positioned in the genome by in situ hybridization to polytene chromosomes. Other sources of STS markers being used for this project are known Drosophila genes and rescued plasmids derived from lethal P element insertions. As of 31 July 1994 we have mapped a total of 1848 STS markers from three classes: P1 end sequences, rescued P elements, and known Drosophila genes. A total of 1299 of these STS markers derive from P1 end sequences. Together these STS markers have assigned over 50% of the clones in the library to contigs, and these contigs cover about 70% of the euchromatin. At the end of the initial grant period (31 July 1995) we are confident that 90-95% of the euchromatic genome will be represented in P1 contigs. This section of the grant proposal is a renewal of this work and describes our approaches to map completion and contig closure. The experiments proposed in this section are organized in three sequential stages. First, the P1 clones remaining after the current production STS mapping phase exhausts the current supply of in situ localized P1 clones used to generate terminal STS markers will be assigned by additional STS mapping experiments. After all the clones in the library have been assigned to contigs, the second level of experiments proposed in this section are aimed at closing apparent gaps between contigs by additional STS mapping experiments. The final stage of map completion will be efforts to close gaps not represented in the master PI library. Closing these statistical gaps will be accomplished by screening additional P1 clones that provide another seven to eight genomic equivalents above and beyond the coverage provided by the master library. Clones to fill gaps remaining after these experiments will be sought by screening libraries constructed with other vector systems such as lambda, cosmid, and YAC.